Device and method for determining the concentration of a substrate

ABSTRACT

A method for determining the concentration of a substrate in a sample solution using an electrode system comprising a working electrode and a counter electrode, both being formed on an electrically insulating base plate, and a reaction layer which contains at least an oxidoreductase and an electron mediator and is formed on the electrode system to electrochemically measure a reduced amount of the electron mediator resulting from enzyme reaction in the reaction layer, wherein a third electrode is formed as an interfering substance detecting electrode somewhere apart from the reaction layer to detect supply of the sample solution on the basis of an electrical change between the counter electrode and the third electrode. A current flowing between the counter electrode and the third electrode is measured which is taken as a positive error. Subsequently, voltage application between the counter electrode and the third electrode is released and a voltage for oxidizing the reduced form electron mediator is applied between the working electrode and the counter electrode to measure a current flowing between the two electrodes. Influences of any interfering substance such as easy-to-oxidize substance are reduced, whereby a highly reliable value of substrate determination can be obtained.

TECHNICAL FIELD

[0001] The present invention relates to a method for performing rapidand high accuracy determination of a substrate in a sample in asimplified manner.

BACKGROUND ART

[0002] As the method for quantitative analysis of sugars such as sucroseand glucose, polarimetry, colorimetry, reductiometry, and methods usinga variety of chromatography have been developed. However, all of thosemethods have poor accuracy because of poor specificity to sugars. Ofthose methods, polarimetry is simple in manipulation but is largelyaffected by the temperature during operation. Therefore, polarimetry isnot a suitable method for ordinary people to carry out determination ofsugars at home or elsewhere in a simplified manner.

[0003] Apropos, various types of biosensor have been developed recentlywhich use specific catalytic actions of enzymes.

[0004] In the following, a method of glucose determination will bedescribed as one example of the method of substrate determination in asample solution. A generally known electrochemical method of glucosedetermination is a method which uses glucose oxidase (EC1.1.3.4;hereinafter abbreviated to GOD) and an oxygen electrode or a hydrogenperoxide electrode (for instance, “Biosensor” ed. by Shuichi Suzuki,Kodansha).

[0005] GOD selectively oxidizes a substrate β-D-glucose toD-glucono-δ-lactone using oxygen as an electron mediator. Oxygen isreduced to hydrogen peroxide in the presence of oxygen in the course ofoxidation reaction by GOD. A decreased amount of oxygen is measured bythe oxygen electrode or, otherwise, an increased amount of hydrogenperoxide is measured by the hydrogen peroxide electrode. The decreasedamount of oxygen or increased amount of hydrogen peroxide isproportional to the glucose content in the sample solution, so thatglucose can be determined based on the decreased amount of oxygen orincreased amount of hydrogen peroxide.

[0006] As can be speculated from the reaction process, this method has adrawback that the measurement result is largely affected by the oxygenconcentration in the sample solution. Furthermore, measurement isimpossible in the absence of oxygen in the sample solution.

[0007] Therefore, a novel type glucose sensor has been developed whichdoes not use oxygen as the electron mediator but uses an organiccompound or a metal complex including potassium ferricyanide, ferrocenederivatives, quinone derivatives, etc. as the electron mediator. Thistype of sensor oxidizes a reduced form electron mediator resulting fromenzyme reaction on the electrode and determines glucose concentrationcontained in the sample solution based on the quantity of oxidationcurrent. The use of such organic compound or metal complex as theelectron mediator in place of oxygen enables formation of a reactionlayer while exactly carrying a known amount of GOD and either of suchelectron mediators in a stabilized state. In this case, since thereaction layer can be integrated in an almost dry state with theelectrode system, a disposable type glucose sensor based on thistechnology has been drawing much attention currently.

[0008] The disposable type glucose sensor facilitates measurement ofglucose concentrations with a measurement device by simple introductionof a sample solution into the sensor detachably connected to themeasurement device. Application of such technic is not limited only toglucose determination and can be extended to determination of othersubstrate contained in the sample solution.

[0009] Measurement using the sensor as described before can determinethe substrate concentration based on a flowing oxidation current valueresulting from oxidation of a reduced form electron mediator on aworking electrode. However, when blood, a fruit juice or something likethat is used as a sample, any easy-to-oxidize substance contained in thesample solution, such as ascorbic acid, uric acid, etc. is concurrentlyoxidized on the working electrode together with the reduced formelectron mediator. Oxidation reaction of such easy-to-oxidize substancemay sometimes affect the measurement result.

[0010] In addition, in the measurement using the sensor as mentionedabove, a reaction producing hydrogen peroxide using dissolved oxygen asan electron mediator may proceed concurrently with the reduction of thecarried electron mediator on the reaction layer. Furthermore, thehydrogen peroxide produced by the reaction reoxidizes the reduced formelectron mediator. This may eventually produce a negative error in themeasurement result due to the dissolved oxygen when the substrateconcentration is to be measured based on the oxidation current of thereduced form electron mediator.

[0011] The above-mentioned method often applies a voltage between theworking electrode and a counter electrode to detect liquid junction,namely, to detect supply of sample solution on the basis of anelectrical change between the two electrodes prior to application of avoltage between the working electrode and the counter electrode in orderto obtain a current response. At that time, it sometimes occurs thatmeasurement starts before supply of sufficient amounts of samplesolution to the electrode system due to a change in resistance valuebetween the above-mentioned working electrode and the counter electrode,which may sometimes affect the measurement result. Induction of a changein the condition of an interface of the working electrode may alsoaffect the measurement result.

[0012] Furthermore, a measurement method with a two-electrode systemuses a counter electrode as a reference electrode. This causes a changein potential of the counter electrode as the standard in associationwith the oxidation-reduction reaction at the working electrode, whichalso affects the measurement result.

[0013] The object of the present invention is to eliminateinconveniences as described above and provide a method of determinationfacilitating accurate measurement of substrate concentration by removinginfluences of easy-to oxidize substances.

[0014] Another object of the present invention is to provide a method ofsubstrate determination with lesser variations in sensor response.

DISCLOSURE OF INVENTION

[0015] The present invention is a method for determining theconcentration of a substrate in a sample solution using a biosensorcomprising an electrically insulating base plate, an electrode systemhaving a working electrode, a counter electrode and a third electrode tobe used as an interfering substance detecting electrode, each beingformed on the above-mentioned base plate, and a reaction layer whichcontains at least an oxidoreductase and an electron mediator and isformed on the electrode system omitting the third electrode, wherein theelectron mediator is reduced by the generating electrons upon reactionbetween the substrate contained in the sample solution and theoxidoreductase to measure a reduced amount of the electron mediatorelectrochemically,

[0016] the method being characterized by comprising the following steps:

[0017] (a) a step of applying a voltage between the counter electrodeand the third electrode;

[0018] (b) a step of supplying the sample solution to the reactionlayer;

[0019] (c) a step of detecting an electrical change between the counterelectrode and the third electrode due to supply of the sample solutionto the reaction layer;

[0020] (d) a step of measuring a current flowing between the counterelectrode and the third electrode after the above-mentioned detectingstep (c);

[0021] (e) a step of releasing voltage application between the counterelectrode and the third electrode after the above-mentioned measuringstep (d);

[0022] (f) a step of applying a voltage between the working electrodeand the counter electrode; and

[0023] (g) a step of subsequently measuring a current flowing betweenthe counter electrode and the working electrode.

[0024] The present invention also provides a method for determining theconcentration of a substrate in a sample solution using a biosensorcomprising an electrically insulating base plate, an electrode systemhaving a working electrode, a counter electrode and a third electrode tobe used as an interfering substance detecting electrode, each beingformed on the above-mentioned base plate, a reaction layer whichcontains at least an oxidoreductase and an electron mediator and isformed on the electrode system omitting the third electrode, and a covermember forming a sample solution supply pathway to introduce a samplesolution from a sample solution supply port into the above-mentionedreaction layer on the above-mentioned base plate, the third electrodebeing located upstream of the sample solution supply pathway from thereaction layer, wherein the electron mediator is reduced by the producedelectrons upon reaction between the substrate contained in the samplesolution and the oxidoreductase to measure a reduced amount of theelectron mediator electrochemically,

[0025] the method comprising the following steps:

[0026] (a) a step of applying a voltage between the counter electrodeand the third electrode;

[0027] (b) a step of supplying the sample solution to the reactionlayer;

[0028] (c) a step of detecting an electrical change between the counterelectrode and the third electrode due to supply of the sample solutionto the reaction layer;

[0029] (d) a step of measuring a current flowing between the counterelectrode and the third electrode after the above-mentioned detectingstep (c);

[0030] (e) a step of releasing voltage application between the counterelectrode and the third electrode after the above-mentioned measuringstep (d);

[0031] (f) a step of applying a voltage between the working electrodeand the counter electrode; and

[0032] (g) a step of subsequently measuring a current flowing betweenthe counter electrode and the working electrode.

[0033] For the method of determination in accordance with the presentinvention, the use of the third electrode as reference electrode ispreferred. Namely, a voltage is also applied between the workingelectrode and the third electrode during the above-mentioned step (f).

[0034] When a biosensor with the cover member being integrally combinedwith the above-mentioned base plate is used, it is also preferable toprovide a lecithin carrying layer on an exposed wall surface of thecover member to the sample solution supply pathway.

[0035] It is preferred that the above-mentioned reaction layer furthercontains a hydrophilic polymer.

BRIEF DESCRIPTION OF DRAWINGS

[0036]FIG. 1 is a plan view illustrating a glucose sensor in accordancewith one example of the present invention from which the reaction layerhas been omitted.

[0037]FIG. 2 is an exploded perspective view illustrating a glucosesensor in accordance with another example of the present invention fromwhich the reaction layer has been omitted.

BEST MODE FOR CARRYING OUT THE INVENTION

[0038] A structure of the biosensor to be used in the method ofdetermination in accordance with the present invention will bedescribed.

[0039] First, a first type biosensor will be described by way of FIG. 1.

[0040] In this sensor, a counter electrode 6, a working electrode 7 anda third electrode 8 are formed on an insulating base plate 1 made ofpolyethylene terephthalate, together with respective leads 2, 3 and 4being electrically connected to them. A carbon layer 9 which is formedto facilitate production of reaction layer does not function as anelectrode. A round reaction layer (not shown) containing anoxidoreductase and an electron mediator is formed on the counterelectrode 6, the working electrode 7 and the carbon layer 9 omitting thethird electrode 8. In the figure, numeral 5 represents an insulatinglayer.

[0041] Next, a second type biosensor will be described by way of FIG. 2.

[0042] This sensor is a combination of the base plate 1 in FIG. 1 with acover member comprising a cover 10 and a spacer 11. They are bonded toeach other in a positional relationship as shown by the dotted chainline in FIG. 2 to form a sensor.

[0043] A slit 12 for forming the sample solution supply pathway isformed on the spacer 11, and an air vent 13 is formed on the cover 10.Laminating the cover 11 on the base plate 1 via the spacer 11 to bondthem to each other results in formation of a cavity which serves as thesample solution supply pathway at the slit 12 on the spacer 11 by thebase plate 1, spacer 11 and the cover 10. An end edge of this cavitycommunicates with the air vent 13.

[0044] In this biosensor, the working electrode 7 is located at aposition closer to a sample solution supply port 12 a (corresponding toan open edge of the slit 12) than the semilunar counter electrode 6, andthe third electrode 8 is located at a position still closer to thesample solution supply port 12 a than the working electrode 7. Each ofthese electrodes 6, 7 and 8 is exposed to the above-mentioned cavity.

[0045] In measuring the substrate concentration using theabove-mentioned biosensor, an end portion of the sensor to which theleads 2, 3 and 4 are provided is set on a measurement device first,followed by application of a predetermined potential onto the thirdelectrode 8 with reference to the counter electrode 6. With thepotential being applied, a sample solution containing, for instance,ascorbic acid as an interfering substance is dropped on the reactionlayer to dissolve the reaction layer in the sample solution.

[0046] Upon supply of the sample solution, a liquid supply detectingsystem starts to operate based on an electrical change between thecounter electrode 6 and the third electrode 8 of the electrode system,which in turn starts a measurement timer. At that time, the potential iskept applied between the counter electrode 6 and the third electrode 8,and a current value between the counter electrode 6 and the thirdelectrode 8 is measured when a certain time has passed after detectionof supply of the sample solution. Since the reaction layer is omittedfrom the third electrode 8, it takes slight time until the reduced formelectron mediator resulting from enzyme reaction reaches near the thirdelectrode 8. Therefore, the above-mentioned current value must bederived from the oxidation reaction of the ascorbic acid contained as aninterfering substance.

[0047] Next, the voltage application between the counter electrode 6 andthe third electrode 8 is released.

[0048] Subsequently, a potential for oxidizing the above-mentionedreduced form electron mediator is applied onto the working electrode 7with reference to the counter electrode 6 to measure a current valuebetween the counter electrode 6 and the working electrode 7. Thiscurrent is derived from the oxidation reactions of the reduced formelectron mediator and the preexisting interfering substance ascorbicacid. In other words, the ascorbic acid produces a positive error in themeasurement result. The above-mentioned current value between thecounter electrode 6 and the third electrode 8 mainly reflects only theconcentration of ascorbic acid, so that correction of the measurementresult on the basis of this current value removes any influence ofascorbic acid, whereby an exact substrate concentration can bedetermined.

[0049] The second type sensor detects supply of the sample solutionbetween the counter electrode 6 and the third electrode 8, so that theentire exposed area of the working electrode 7 is filled with the samplesolution with security. As a result, supply of the sample solution canbe determined more reliably.

[0050] In the following, the present invention will be described morespecifically by way of examples.

EXAMPLE 1

[0051] A method of glucose determination will be described. The baseplate shown in FIG. 1 was used for the base plate of a glucose sensor.The glucose sensor was produced as follows.

[0052] A silver paste was printed on the insulating base plate 1 made ofpolyethylene terephthalate by using screen printing to form therespective leads 2, 3 and 4. Next, a conductive carbon paste containinga resin binder was further printed on the base plate 1 to from thecounter electrode 6, the working electrode 7, the third electrode 8 andthe carbon layer 9. The counter electrode 6, the working electrode 7 andthe third electrode 8 are electrically connected to the leads 2, 3 and4, respectively.

[0053] Then, an insulating paste was printed on the base plate 1 to formthe insulating layer 5. The insulating layer 5 covers a periphery ofeach of the counter electrode 6, the working electrode 7, the thirdelectrode 8 and the carbon layer 9, whereby an exposed area of each ofthe counter electrode 6, the working electrode 7, the third electrode 8and the carbon layer 9 is held constant. The insulating layer 5partially covers the leads 2, 3 and 4.

[0054] Next, an aqueous solution of carboxymethyl cellulose (hereinafterabbreviated to CMC) was dropped on the counter electrode 6, workingelectrode 7 and carbon layer 9 omitting the third electrode 8 and driedto form a CMC layer. Dropping an aqueous solution containing GOD as anenzyme and potassium ferricyanide as an electron mediator on the CMClayer once dissolves the CMC layer composed of a hydrophilic polymer,which is formed into a reaction layer by the subsequent drying process,with CMC being mixed with the enzyme and the other constituent. However,the absence of agitation, etc. results in incomplete mixing of the both,whereby the surface of the electrode system is covered with only CMC. Inother words, because of no contact of the enzyme and the electronmediator with the surface of the electrode system, adsorption of proteinonto the surface of the electrode system can be prevented.

[0055] In order to measure glucose concentrations using this sensor, anend portion of the sensor at which the leads 2, 3 and 4 are provided wasset to a measurement device and a potential of 500 mV was applied ontothe third electrode 8 with reference to the counter electrode 6. Withthe potential being kept applied, an aqueous glucose solution containingascorbic acid as an interfering substance was dropped on the reactionlayer as a sample solution at 30 μl. The reaction layer on the electrodesystem dissolved in the dropped sample solution.

[0056] Upon supply of the sample solution, a liquid supply detectingsystem started to operate based on an electrical change between thecounter electrode 6 and the third electrode 8 of the electrode system.This started a measurement timer. At that time, the potentialapplication is being continued between the counter electrode 6 and thethird electrode 8, and after a lapse of a certain time from thedetection of supply of the sample solution, a current between thecounter electrode 6 and the third electrode 8 was measured. The currentwas derived from the oxidation reaction of the ascorbic acid containedas an interfering substance and had a proportional relationship with itsconcentration. After measurement of the current between the counterelectrode 6 and the third electrode 8, the voltage application betweenthe both electrodes was released.

[0057] As mentioned above, the reaction layer was not disposed on thethird electrode 8. Therefore, it takes slight time until arrival offerrocyanide ions resulting from enzyme reaction near the thirdelectrode 8. Namely, the current value between the counter electrode 6and the third electrode 8 during an interval until arrival offerrocyanide ions mainly reflects only the concentration of ascorbicacid.

[0058] Furthermore, 25 seconds after detection of sample solution, 500mV was applied onto the working electrode 7 with reference to thecounter electrode 6 and a current value between the counter electrode 6and the working electrode 7 was measured after 5 seconds.

[0059] Reaction of ferricyanide ions, glucose and GOD in the solutioneventually oxidizes glucose to gluconolactone and reduces ferricyanideions to ferrocyanide ions. The concentration of ferrocyanide ion isproportional to the glucose concentration. A current between the counterelectrode 6 and the working electrode 7 after 30 seconds of detection ofthe sample solution is derived from the oxidation reactions offerrocyanide ions and preexisting ascorbic acid. This means thatascorbic acid produces a positive error in the measurement result.However, as described before, the current value between the counterelectrode 6 and the third electrode 8 mainly reflects only theconcentration of ascorbic acid. Therefore, correction of the measurementresult based on that result can remove any effects of ascorbic acidthereby enabling determination of accurate glucose concentration.

EXAMPLE 2

[0060] The electrodes 6, 7, 8 and carbon layer 9 were formed on the baseplate 1 in the same manner as in Example 1. Next, an aqueous CMCsolution was dropped on the counter electrode 6, working electrode 7 andcarbon layer 9 while omitting the third electrode 8 and dried to formthe CMC layer, on which an aqueous solution containing GOD as an enzymeand potassium ferricyanide as an electron mediator was further droppedand dried to form the reaction layer.

[0061] Next, for further smoothing supply of the sample solution to thereaction layer, an organic solvent solution of lecithin, such as toluenesolution, for example, was spread from the sample solution supply porttoward the reaction layer and dried to form a lecithin layer on thereaction layer. Next, the cover 10 and the spacer 11 were bonded to thebase plate 1 in a positional relationship as shown by the dotted chainline in FIG. 2 to form a glucose sensor.

[0062] The sensor was set on a measurement device and a potential of 500mV was applied onto the third electrode 8 with reference to the counterelectrode 6. With the potential kept applied, an aqueous glucosesolution containing ascorbic acid as an interfering substance wassupplied through the sample solution supply port 12 a at 3 μl as asample solution. The sample solution reached the air vent 13 by passingthrough the sample solution supply pathway and dissolved the reactionlayer on the electrode system.

[0063] Upon supply of the sample solution, a liquid supply detectingsystem started to operate based on an electrical change between thecounter electrode 6 and the third electrode 8 of the electrode system.This started a measurement timer. At that time, the potentialapplication is being continued between the counter electrode 6 and thethird electrode 8, and after a lapse of a certain time from thedetection of supply of the sample solution, a current between thecounter electrode 6 and the third electrode 8 was measured. The currentwas derived from the oxidation reaction of the ascorbic acid containedas an interfering substance and had a proportional relationship with itsconcentration. After measurement of the current between the counterelectrode 6 and the third electrode 8, the voltage application betweenthe two electrodes was released.

[0064] As mentioned above, the reaction layer was not disposed on thethird electrode 8. Therefore, it takes slight time until arrival offerrocyanide ions resulting from enzyme reaction near the thirdelectrode 8. Namely, the current value between the counter electrode 6and the third electrode 8 during an interval until arrival offerrocyanide ion mainly reflects only the concentration of ascorbicacid.

[0065] Furthermore, 25 seconds after detection of the sample solution,500 mV was applied onto the working electrode 7 with reference to thecounter electrode 6 and a current value between the counter electrode 6and the working electrode 7 was measured after 5 seconds.

[0066] Reaction of ferricyanide ions, glucose and GOD in the solutioneventually oxidizes glucose to gluconolactone and reduces ferricyanideions to ferrocyanide ions. The concentration of ferrocyanide ion isproportional to the glucose concentration. A current between the counterelectrode 6 and the working electrode 7 after 30 seconds of detection ofthe sample solution is derived from the oxidation reactions offerrocyanide ions and preexisting ascorbic acid. This means thatascorbic acid produces a positive error in the measurement result.However, as described before, the current value between the counterelectrode 6 and the third electrode 8 mainly reflects only theconcentration of ascorbic acid. Therefore, correction of the measurementresult based on that result can remove any effects of ascorbic acidthereby enabling determination of accurate glucose concentration.

[0067] In the present example, due to detection of supply of the samplesolution between the counter electrode 6 and the third electrode 8, theentire exposed portion of the working electrode 7 is filled with thesample solution with security. This enables still more reliabledetermination of supply of the sample solution.

EXAMPLE 3

[0068] A glucose sensor was produced in the same manner as in Example 2.

[0069] The sensor was set on a measurement device and a potential of 500mV was applied onto the third electrode 8 with reference to the counterelectrode 6. With the potential kept applied, an aqueous glucosesolution containing ascorbic acid as an interfering substance wassupplied through the sample solution supply port 12 a at 3 μl as asample solution. The sample solution reached the air vent 13 by passingthrough the sample solution supply pathway and dissolved the reactionlayer on the electrode system.

[0070] Upon supply of the sample solution, a liquid supply detectingsystem started to operate based on an electrical change between thecounter electrode 6 and the third electrode 8 of the electrode system,which then started a measurement timer. At that time, the potentialapplication is being continued between the counter electrode 6 and thethird electrode 8, and after a lapse of a certain time from thedetection of supply of the sample solution, a current between thecounter electrode 6 and the third electrode 8 was measured. The currentwas derived from the oxidation reaction of the ascorbic acid containedas an interfering substance and had a proportional relationship with itsconcentration. After measurement of the current between the counterelectrode 6 and the third electrode 8, the voltage application betweenthe two electrodes was released.

[0071] As mentioned above, the reaction layer was not disposed on thethird electrode 8. Therefore, it takes slight time until arrival offerrocyanide ions resulting from enzyme reaction near the thirdelectrode 8. Namely, the current value between the counter electrode 6and the third electrode 8 during an interval until arrival offerrocyanide ions mainly reflects only the concentration of ascorbicacid.

[0072] Furthermore, 25 seconds after detection of the sample solution,500 mV was applied onto the working electrode 7 with reference to thethird electrode 8 and a current value between the counter electrode 6and the working electrode 7 was measured after 5 seconds.

[0073] Reaction of ferricyanide ions, glucose and GOD in the solutioneventually oxidizes glucose to gluconolactone and reduces ferricyanideions to ferrocyanide ions. The concentration of ferrocyanide ion isproportional to the glucose concentration. A current between the counterelectrode 6 and the working electrode 7 after 30 seconds of detection ofthe sample solution is derived from the oxidation reactions offerrocyanide ions and preexisting ascorbic acid. This means thatascorbic acid produces a positive error in the measurement result.However, as described before, the current value between the counterelectrode 6 and the third electrode 8 mainly reflects only theconcentration of ascorbic acid. Therefore, correction of the measurementresult based on that result can remove any effects of ascorbic acidthereby enabling determination of accurate glucose concentration.

[0074] Additional measurement of a potential of the third electrode 8during potential application onto the working electrode 7 with referenceto a silver/silver chloride electrode demonstrated almost no change inpotential of the third electrode 8 although oxidation reaction occurredat the working electrode 7. Variations in sensor response were alsodecreased compared to the conventional method which detects liquidjunction based on a change in resistance between the working electrodeand the counter electrode.

EXAMPLE 4

[0075] In the same manner as in Example 2, the reaction layer was formedon the counter electrode 6, working electrode 7 and carbon layer 9 whileomitting the third electrode 8.

[0076] Next, an organic solvent solution of lecithin such as toluenesolution, for example, was spread on a groove formed on the cover memberfor forming the sample solution supply pathway and dried, thereby toform the lecithin layer for the purpose of still more smoothing supplyof the sample solution to the reaction layer. Then, the cover 10 and thespacer 11 were bonded to the base plate 1 in a positional relationshipas shown by the dotted chain line in FIG. 2, which gave a glucosesensor.

[0077] Positioning the lecithin layer from the reaction layer over thethird electrode 8 may sometimes increase variations in sensor responsecharacteristics due to a change of the surface of the third electrode bythe lecithin layer. Positioning the lecithin layer on the cover memberside as shown above resulted in a decrease in such variations, and theresponse characteristics improved.

EXAMPLE 5

[0078] A glucose sensor was produced completely in the same manner as inExample 2 except for omission of the CMC layer from the reaction layer.

[0079] And, the result of measurement in the same manner as in Example 2showed dependency on the ascorbic acid and glucose concentrationsdespite increased variations in sensor response as compared to the caseof including the CMC layer.

EXAMPLE 6

[0080] A glucose sensor was produced in the same manner as in Example 4.

[0081] The sensor was set on a measurement device and a potential of−1,300 mV was applied onto the third electrode 8 with reference to thecounter electrode 6. With the potential kept applied, an air saturatedaqueous glucose solution was supplied through the sample solution supplyport 12 a at 3 μl as a sample solution. The sample solution reached theair vent 13 by passing through the sample solution supply pathway anddissolved the reaction layer on the electrode system.

[0082] Upon supply of the sample solution, a liquid supply detectingsystem started to operate based on an electrical change between thecounter electrode 6 and the third electrode 8 of the electrode system,which started a measurement timer. At that time, the potentialapplication is being continued between the counter electrode 6 and thethird electrode 8, and after a lapse of a certain time from thedetection of supply of the sample solution, a current between thecounter electrode 6 and the third electrode 8 was measured. The currentwas derived from the reduction reaction of the dissolved oxygen. When aglucose solution degassed with argon was supplied, the reduction currentdecreased drastically. After measurement of the current between thecounter electrode 6 and the third electrode 8, the voltage applicationbetween the two electrodes was released.

[0083] As mentioned above, the reaction layer was not disposed on thethird electrode 8. Therefore, it takes slight time until arrival offerricyanide ions in the reaction layer near the third electrode 8.Namely, the current value between the counter electrode 6 and the thirdelectrode 8 during an interval until arrival of ferricyanide ions mainlyreflects only the concentration of dissolved oxygen.

[0084] Furthermore, 25 seconds after detection of the sample solution,500 mV was applied onto the working electrode 7 with reference to thethird electrode 8 and a current value between the counter electrode 6and the working electrode 7 was measured after 5 seconds.

[0085] Reaction of ferricyanide ions, glucose and GOD in the solutioneventually oxidizes glucose to gluconolactone, and reduction offerricyanide ions to ferrocyanide ions occurs with this oxidationreaction.

[0086] On the other hand, a reaction proceeds at the same time as acompetitive reaction where dissolved oxygen is reduced to hydrogenperoxide as the glucose is oxidized to gluconolactone due to the actionof the dissolved oxygen in the sample solution as an electron mediator.Hydrogen peroxide generating by this reaction reoxidizes ferrocyanideions to ferricyanide ions. Therefore, if glucose concentration is to bemeasured based on an oxidation current of ferrocyanide ion, suchdissolved oxygen can produce a negative error in the measurement result.

[0087] However, as mentioned before, the current value between thecounter electrode 6 and the third electrode 8 mainly reflects only theconcentration of dissolved oxygen. Therefore, correction of themeasurement result based on that result can remove any effects ofdissolved oxygen thereby enabling determination of accurate glucoseconcentration.

EXAMPLE 7

[0088] A glucose sensor was produced in the same manner as in Example 4.

[0089] The sensor was set on a measurement device and a potential of 500mV was applied onto the third electrode 8 with reference to the counterelectrode 6. With the potential kept applied, an aqueous glucosesolution containing ascorbic acid as an interfering substance wassupplied through the sample solution supply port 12 a at 3 μl as asample solution. The sample solution reached the air vent 13 by passingthrough the sample solution supply pathway and dissolved the reactionlayer on the electrode system.

[0090] Upon supply of the sample solution, a liquid supply detectingsystem started to operate based on an electrical change between thecounter electrode 6 and the third electrode 8 of the electrode system,which started a measurement timer. At that time, the potentialapplication is being continued between the counter electrode 6 and thethird electrode 8. Two seconds after detection of supply of the samplesolution, the potential to be applied onto the third electrode 8 waschanged to −1,300 mV. The current between the counter electrode 6 andthe third electrode 8 was measured at two time points immediately beforeand 3 seconds after the potential change to −1,300 mV. The currentimmediately before the potential change is mainly dependent on theconcentration of ascorbic acid. On the other hand, the current 3 secondsafter the potential change to −1,300 mV is mainly dependent on theconcentration of dissolved oxygen in the sample solution.

[0091] After measurements of the current between the counter electrode 6and the third electrode 8 after 2 and 5 seconds of supply of the samplesolution, the voltage application between the two electrodes wasreleased.

[0092] Twenty-five seconds after detection of the sample solution, 500mV was further applied onto the working electrode 7 with reference tothe third electrode 8 and the current between the counter electrode 6and the working electrode 7 was measured after 5 seconds.

[0093] As described above, the current value between the counterelectrode 6 and the third electrode 8 mainly reflects concentrations ofascorbic acid and dissolved oxygen. Therefore, concentrations of thosetwo substances can be determined based on that current value. Therefore,correction of the measurement result based on that result can remove anyeffects of ascorbic acid and dissolved oxygen thereby enablingdetermination of accurate glucose concentration.

[0094] In the foregoing examples, although the potential to be appliedonto the third electrode 8 for sensing supply of the sample solution todetect ascorbic acid or dissolved oxygen was 500 mV or −1,300 mV, thepresent invention is not limited to those potential values. Moreover,although a potential of 500 mV was applied onto the working electrode 7to obtain a response current, the present invention is not limited tothis potential value and any potential may be used if it can oxidize thereduced form electron mediator resulting from a series of reaction. Thetime point to measure the current value is also not limited to thoseused in the foregoing examples.

[0095] In the foregoing examples, although carboxymethyl cellulose wasused as the hydrophilic polymer, a variety of hydrophilic polymers canbe used for forming the hydrophilic polymer layer. Exemplary hydrophilicpolymers include hydroxyethyl cellulose, hydroxypropyl cellulose, methylcellulose, ethyl cellulose, ethylhydroxyethyl cellulose,carboxymethylethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol,polyamino acid such as polylysin, polystyrene sulfonate, gelatin and itsderivatives, polyacrylic acid and its salts, polymethacrylic acid andits salts, starch and its derivatives, and a polymer of maleic anhydrideor a maleate. Of them, carboxymethyl cellulose, hydroxyethyl celluloseand hydroxypropyl cellulose are preferred.

[0096] The oxidoreductase to be contained in the reaction layer isselected depending on the substrate contained in the sample solution.Exemplary oxidoreductases include fructose dehydrogenase, glucoseoxidase, alcohol oxidase, lactate oxidase, cholesterol oxidase, xanthineoxidase, and amino-acid oxidase.

[0097] As the electron mediator, potassium ferricyanide, p-benzoquinone,phenazine methosulfate, methylene blue, and ferrocene derivatives may beexemplified. These electron mediators may be used singly or incombination of two or more.

[0098] The above-exemplified enzymes and electron mediators may bedissolved in the sample solution or may be prevented from dissolving inthe sample solution by fixing the reaction layer onto the base plate andso on. When the enzyme and the electron mediator are to be fixed, thereaction layer preferably contains the hydrophilic polymer.

[0099] In the foregoing examples, specific electrode systems were shown,but the present invention is not limited to those electrode systems withrespect to the shape of the electrode and location of the electrodes andleads.

[0100] In the foregoing examples, although carbon was used as thematerial of the third electrode, the present invention is not limited tocarbon electrode and those made of other conductive material or asilver/silver chloride electrode can also be used.

[0101] Industrial Applicability

[0102] As discussed above, the present invention enables substratedetermination of high reliability.

1. A method for determining the concentration of a substrate in a samplesolution using a biosensor comprising an electrically insulating baseplate, an electrode system having a working electrode, a counterelectrode and a third electrode to be used as an interfering substancedetecting electrode, each being formed on said base plate, and areaction layer which contains at least an oxidoreductase and an electronmediator and is formed on the electrode system omitting the thirdelectrode, wherein said electron mediator is reduced by the producedelectrons upon reaction between the substrate contained in the samplesolution and the oxidoreductase to measure a reduced amount of saidelectron mediator electrochemically, said method being characterized bycomprising: (a) a step of applying a voltage between the counterelectrode and the third electrode; (b) a step of supplying the samplesolution to the reaction layer; (c) a step of detecting an electricalchange between the counter electrode and the third electrode due tosupply of the sample solution to the reaction layer; (d) a step ofmeasuring a current flowing between the counter electrode and the thirdelectrode after said detecting step (c); (e) a step of releasing voltageapplication between the counter electrode and the third electrode aftersaid measuring step (d); (f) a step of applying a voltage between theworking electrode and the counter electrode; and (g) a step ofsubsequently measuring a current flowing between the counter electrodeand the working electrode.
 2. The method for determining substrate inaccordance with claim 1, wherein said step (f) also applies a voltagebetween the working electrode and the third electrode.
 3. A method fordetermining the concentration of a substrate in a sample solution usinga biosensor comprising an electrically insulating base plate, anelectrode system having a working electrode, a counter electrode and athird electrode to be used as an interfering substance detectingelectrode, each being formed on said base plate, a reaction layer whichcontains at least an oxidoreductase and an electron mediator and isformed on said electrode system omitting the third electrode, and acover member forming a sample solution supply pathway to introduce asample solution from a sample solution supply port into said reactionlayer on said base plate, said third electrode being located upstream ofsaid sample solution supply pathway from said reaction layer, whereinsaid electron mediator is reduced by the produced electrons uponreaction between the substrate contained in the sample solution and theoxidoreductase to measure a reduced amount of said electron mediatorelectrochemically, said method being characterized by comprising: (a) astep of applying a voltage between the counter electrode and the thirdelectrode; (b) a step of supplying the sample solution to the reactionlayer; (c) a step of detecting an electrical change between the counterelectrode and the third electrode due to supply of the sample solutionto the reaction layer; (d) a step of measuring a current flowing betweenthe counter electrode and the third electrode after said detecting step(c); (e) a step of releasing voltage application between the counterelectrode and the third electrode after said measuring step (d); (f) astep of applying a voltage between the working electrode and the counterelectrode; and (g) a step of subsequently measuring a current flowingbetween the counter electrode and the working electrode.
 4. The methodfor determining substrate in accordance with claim 3, wherein said step(f) also applies a voltage between the working electrode and the thirdelectrode.
 5. The method for determining substrate in accordance withclaim 3, wherein a biosensor disposed with a layer essentially composedof lecithin on an exposed surface of the sample solution supply pathwayof said cover member is used.
 6. The method for determining substrate inaccordance with claim 1, wherein a biosensor further containing ahydrophilic polymer in said reaction layer is used.
 7. The method fordetermining substrate in accordance with claim 3, wherein a biosensorfurther containing a hydrophilic polymer in said reaction layer is used.